Several techniques are currently available for detection and typing of bacterial and viral pathogens. This includes methods employing:                1) indirect determination of genetic sequence including species/strain-specific PCR, repetitive sequence-based PCR (rep-PCR), pulse-field gel electrophoresis (PFGE), and optical DNA mapping,        2) direct determination of the sequence by either multi-locus sequence typing (MLST) or whole bacterial genome sequencing, or        3) a combination of the above such as determination of the base composition of PCR products by mass spectrometry.        
The first group of techniques (Cepheid, PathoGenetix, Inc., OpGen, Inc.) has lower resolution and discrimination power compared to the second group of techniques and is limited by the small number of conserved genomic regions interrogated. The techniques in the second group, however, have been prohibitively expensive and provide only low throughput, although, with the introduction of 2nd (SOLiD and PGM by Life Technologies/Ion Torrent, Illumina, Roche 454, Complete Genomics) and 3rd (Pacific Biosciences, Helicos) generation high throughput sequencing techniques, the per sample cost is trending below $10,000. Moreover, the currently available sequencing technologies suffer from either complex sample preparation and DNA cluster generation (SOLiD and IonTorrent by Life Technologies, Roche 454, Complete Genomics, Illumina), short read length (Helicos, SOLiD, Illumina), or high error rate (Pacific Biosciences). Additionally, the currently available single-molecule sequencing instruments (Pacific Biosciences and Helicos) are bulky, very expensive, and require highly trained personnel to operate. The third group of techniques (Ibis Biosciences Inc.) is able to determine the nucleotide composition of only relatively short sequences of PCR products and suffers from all limitations of conventional PCR.
In contrast to currently available single molecule technologies (Helicos, Pacific Biosciences), the rotation-dependent transcriptional sequencing described herein does not require development of mutant polymerases capable of incorporating modified nucleotides, expensive labeled nucleotides, or lasers and costly high-speed cameras. Thus, the rotation-dependent transcriptional sequencing described herein can be integrated into inexpensive portable point-of-care systems.
In addition, the rotation-dependent transcriptional sequencing described herein allows for ultimate flexibility and fast reconfiguration; permitting rapid response to unforeseen endemic threats, emerging diseases, pandemics and new bioterror threats by simply updating the platform-associated nucleic acid database and software without any change of the reagents. This is in contrast to numerous diagnostic platforms exploiting PCR, where significant time is required for assay reconfiguration and validation before platform redeployment to address any new targets. This is also in contrast to non-sequencing single-molecule Genome Sequence Scanning platform using Direct Linear Analysis (DLA) technology (PathoGenetiX), which relies upon a spatial pattern of tags separated by at least 3 kb and makes this technology insensitive to sub-kb insertions or deletions as well as single-nucleotide variances.
Furthermore, the rotation-dependent transcriptional sequencing described herein allows for ultimate multiplexing capability. While PCR- and microarray-based methods are limited by the detection of only known infectious agent(s) and cannot identify variants that are mutated or bioengineered (i.e. with a single nucleotide difference), the rotation-dependent transcriptional sequencing described herein is, in a sense, “target agnostic,” as the methods decode primary structure of any and all DNA molecules in or extracted from the specimen, and, thus, is capable of detecting and identifying thousands of known or unknown (e.g., genetically-modified) targets simultaneously. Such an inherited “broadband” multiplexing capability provided by the systems and methods described herein is in contrast to approaches employing PCR that require specific sets of reagents (primers and probes) for detection of each pathogen. Additionally, PCR-based technologies are limited by the number of assays allowed in multiplexed reactions simply due to the nature of PCR, or by the need to split the sample (i.e., containing the target nucleic acids) between multiple reactions, thereby compromising the sensitivity of detection and the accuracy of quantification.